Microcystin-LR heterologous genetically engineered antibody recombinant and its binding activity improvement and...

Microcystin-LR (MC-LR) is a high-toxic biohazard that pollutes ecological environment and agroproducts. In this study, a newly recombined genetically engineered antibody (AV_HH-MV_H) with higher thermal stability and binding activity was designed by chain shuffling and based on our previously obtained anti-MC-LR scFv and nanobody. Based on AV_HH-MV_H template, a capacity of 8.99 × 10⁵ CFU/mL of phage display AV_HH-MV_H mutagenesis library was constructed by site-directed mutagenesis in MV_H-CDR3 region, and then used for ultrasensitive mutants screening. Afterwards, a total of five positive AV_HH-MV_H mutants were isolated from the mutagenesis library, and their binding activity was higher than AV_HH-MV_H for MC-LR. The AV_HH-MV_H mutant 3 was cloned into pET-25b vector for soluble expression, and the concentration of target protein expressed in culture system was 43.5 mg/L. An indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) was established based on purified AV_HH-MV_H mutant 3 protein, and it showed ultrasensitive binding activity for MC-LR with the detection limit of 0.0075 μg/L, which was far below the maximum residue limit standard of 1.0 μg/L in drinking water proposed by World Health Organization. The established IC-ELISA shows good accuracy, repeatability, stability and applicability for MC-LR spiked samples, and it is promising for MC-LR ultrasensitive monitoring.